Polla Hergert - Academia.edu (original) (raw)

Papers by Polla Hergert

Research paper thumbnail of Transforming Growth Factor-β1 Induced Epithelial-Mesenchymal Transition in Hepatic Fibrosis

Hepatogastroenterology, 2011

The epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) are cruci... more The epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) are crucial for the regulation of cellular plasticity during liver fibrosis. Transforming growth factor (TGF)-β1 is an important cytokine for the induction of the EMT in liver fibrosis. TGF-β1 signaling induces the EMT through various signaling mechanisms and is the predominant agent mediating these fibrotic changes. Chronic exposure to TGF-β1 induces the transition of hepatocytes to collagen-producing mesenchymal cells, prolonged exposure of hepatocytes to TGF-β1 increases the expression of collagen and induces cytoskeletal rearrangement that resembles the EMT. These morphological and molecular alterations may provide the foundation for liver fibrosis. This review discussed the relation and mechanisms between EMT and liver fibrosis and ulteriorly elaborated on TGF-β1 induced EMT and each of their roles in liver fibrosis. Better understanding of the cellular and molecular characteristics of the cirrhotic hepatocyte may enable the development of chemo-preventative agents for liver fibrosis.

Research paper thumbnail of Identification of a Cell-of-Origin for Fibroblasts Comprising the Fibrotic Reticulum in Idiopathic Pulmonary Fibrosis

The American Journal of Pathology, 2014

Research paper thumbnail of Statistical Profiles of PTK1 Kinetochore Microtubule Plus-End Conformations Using High-Throughput Electron Tomography

Microscopy and Microanalysis, 2004

Extended abstract of a paper presented at Microscopy and Microanalysis 2004 in Savannah, Georgia,... more Extended abstract of a paper presented at Microscopy and Microanalysis 2004 in Savannah, Georgia, USA, August 1–5, 2004.

Research paper thumbnail of Low α2β1 Integrin Function Enhances the Proliferation of Fibroblasts from Patients with Idiopathic Pulmonary Fibrosis by Activation of the β-Catenin Pathway

The American Journal of Pathology, 2012

Idiopathic pulmonary fibrosis (IPF) is a progressive and incurable fibroproliferative disorder ch... more Idiopathic pulmonary fibrosis (IPF) is a progressive and incurable fibroproliferative disorder characterized by unrelenting proliferation of fibroblasts and their deposition of collagen within alveoli, resulting in permanently scarred, nonfunctional airspaces. Normally, polymerized collagen suppresses fibroblast proliferation and serves as a physiological restraint to limit fibroproliferation after tissue injury. The IPF fibroblast, however, is a pathologically altered cell that has acquired the capacity to elude the proliferation-suppressive effects of polymerized collagen. The mechanism for this phenomenon remains incompletely understood. Here, we demonstrate that expression of ␣ 2 ␤ 1 integrin, a major collagen receptor, is pathologically low in IPF fibroblasts interacting with polymerized collagen. Low integrin expression in IPF fibroblasts is associated with a failure to induce PP2A phosphatase activity, resulting in abnormally high levels of phosphorylated (inactive) GSK-3␤ and high levels of active ␤-catenin in the nucleus. Knockdown of ␤-catenin in IPF fibroblasts inhibits their ability to proliferate on collagen. Interdiction of ␣ 2 ␤ 1 integrin in control fibroblasts reproduces the IPF phenotype and leads to the inability of these cells to activate PP2A, resulting in high levels of phosphorylated GSK-3␤ and active ␤-catenin and in enhanced proliferation on collagen. Our findings indicate that the IPF fibroblast phenotype is characterized by low ␣ 2 ␤ 1 integrin expression, resulting in a failure of integrin to activate PP2A phosphatase, which permits inappropriate activation of the ␤-catenin pathway.

Research paper thumbnail of Pathological Alteration of FoxO3a Activity Promotes Idiopathic Pulmonary Fibrosis Fibroblast Proliferation on Type I Collagen Matrix

The American Journal of Pathology, 2011

Idiopathic pulmonary fibrosis (IPF) is a prevalent, progressive, and incurable fibroproliferative... more Idiopathic pulmonary fibrosis (IPF) is a prevalent, progressive, and incurable fibroproliferative lung disease. The phenotype of IPF fibroblasts is characterized by their ability to elude the proliferation-suppressive properties of polymerized type I collagen. The mechanism underlying this pathological response is incompletely understood but involves aberrant activation of the phosphatidylinositol 3-kinase-Akt signaling pathway owing to inappropriately low phosphatase and tensin homolog phosphatase activity. Akt can phosphorylate and inactivate the forkhead box O3a (FoxO3a) transcriptional factor, which, when transcriptionally active, increases the expression of the CDK inhibitor p27 and promotes cell cycle arrest. Herein, we demonstrate that IPF fibroblasts display high levels of inactive FoxO3a compared with nonfibrotic control fibroblasts because of their high Akt activity. We found that p27 levels are decreased in IPF compared with control fibroblasts cultured on polymerized collagen. Furthermore, overexpression of FoxO3a in IPF fibroblasts increases p27 levels and suppresses the ability of IPF fibroblasts to proliferate on polymerized collagen. In contrast, the expression of dominant-negative FoxO3a augmented control fibroblast proliferation. IHC examination of fibroblastic foci in IPF lung tissue demonstrates the presence of inactive FoxO3a in cells within fibroblastic foci. These data indicate that the ability of IPF fibroblasts to circumvent the proliferation-suppressive properties of polymerized collagen involves inactivation of FoxO3a by high Akt activity, resulting in down-regulation of p27.

Research paper thumbnail of Ultrastructure of Nocodazole-Treated PtK1 Kinetochore after High-Pressure Freezing and Freeze-Substitution

Extended abstract of a paper presented at Microscopy and Microanalysis 2005 in Honolulu, Hawaii, ... more Extended abstract of a paper presented at Microscopy and Microanalysis 2005 in Honolulu, Hawaii, USA, July 31--August 4, 2005

Research paper thumbnail of An insoluble protein matrix is required for centrosome function

Research paper thumbnail of The effects of collagen-rich extracellular matrix on the intracellular delivery of glycol chitosan nanoparticles in human lung fibroblasts

International Journal of Nanomedicine

Recent progress in nanomedicine has shown a strong possibility of targeted therapy for obstinate ... more Recent progress in nanomedicine has shown a strong possibility of targeted therapy for obstinate chronic lung diseases including idiopathic pulmonary fibrosis (IPF). IPF is a fatal lung disease characterized by persistent fibrotic fibroblasts in response to type I collagen-rich extracellular matrix. As a pathological microenvironment is important in understanding the biological behavior of nanoparticles, in vitro cellular uptake of glycol chitosan nanoparticles (CNPs) in human lung fibroblasts was comparatively studied in the presence or absence of type I collagen matrix. Primary human lung fibroblasts from non-IPF and IPF patients (n=6/group) showed significantly increased cellular uptake of CNPs (.33.6-78.1 times) when they were cultured on collagen matrix. To elucidate the underlying mechanism of enhanced cellular delivery of CNPs in lung fibroblasts on collagen, cells were pretreated with chlorpromazine, genistein, and amiloride to inhibit clathrin-mediated endocytosis, caveolae-mediated endocytosis, and macropinocytosis, respectively. Amiloride pretreatment remarkably reduced the cellular uptake of CNPs, suggesting that lung fibroblasts mainly utilize the macropinocytosisdependent mechanism when interacted with collagen. In addition, the internalization of CNPs was predominantly suppressed by a phosphoinositide 3-kinase (PI3K) inhibitor in IPF fibroblasts, indicating that enhanced PI3K activity associated with late-stage macropinocytosis can be particularly important for the enhanced cellular delivery of CNPs in IPF fibroblasts. Our study strongly supports the concept that a pathological microenvironment which surrounds lung fibroblasts has a significant impact on the intracellular delivery of nanoparticles. Based on the property of enhanced intracellular delivery of CNPs when fibroblasts are made to interact with a collagen-rich matrix, we suggest that CNPs may have great potential as a drug-carrier system for targeting fibrotic lung fibroblasts.

Research paper thumbnail of Idiopathic pulmonary fibrosis fibroblasts become resistant to Fas ligand-dependent apoptosis via the alteration of decoy receptor 3

The Journal of pathology, Sep 1, 2016

Idiopathic pulmonary fibrosis (IPF) is an irreversible lethal lung disease with an unknown etiolo... more Idiopathic pulmonary fibrosis (IPF) is an irreversible lethal lung disease with an unknown etiology. IPF patients' lung fibroblasts express inappropriately high Akt activity, protecting them in response to an apoptosis-inducing type I collagen matrix. FasL, a ligand for Fas, is known to be increased in the lung tissues of patients with IPF, implicated with the progression of IPF. Expression of Decoy Receptor3 (DcR3), which binds to FasL, thereby subsequently suppressing the FasL-Fas-dependent apoptotic pathway, is frequently altered in various human disease. However, the role of DcR3 in IPF fibroblasts in regulating their viability has not been examined. We found that enhanced DcR3 expression exists in the majority of IPF fibroblasts on collagen matrices, resulting in the protection of IPF fibroblasts from FasL-induced apoptosis. Abnormally high Akt activity suppresses GSK-3β function, thereby accumulating the nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) in the nuc...

Research paper thumbnail of LC3-2 expression was up-regulated in IPF fibroblasts in the presence of rapamycin on collagen

Research paper thumbnail of Caveolin-1 (Cav-1) Alteration By Aberrant Forkhead Box O3a (FoxO3a) Activity Confers A Highly Proliferative And An Anti-Apoptotic IPF Fibroblast Phenotypes On Type I Collagen Matrix

B66. THE FIBROBLAST POOL ON THE MOVE: DERIVATION AND TARGETING, 2012

Research paper thumbnail of Fibroblasts within the fibroblastic foci of IPF patient lung specimens express inactive FoxO3a and α-smooth muscle actin but not cav-1 and cleaved caspase-3

Research paper thumbnail of Reduced FoxO3a expression causes low autophagy in idiopathic pulmonary fibrosis fibroblasts on collagen matrices

American Journal of Physiology - Lung Cellular and Molecular Physiology, 2015

Idiopathic pulmonary fibrosis (IPF) is a chronic and fatal lung disease, and fibroblasts derived ... more Idiopathic pulmonary fibrosis (IPF) is a chronic and fatal lung disease, and fibroblasts derived from patients with IPF are resistant to type I collagen matrix-induced cell death. The alteration of the PTEN-Akt axis permits IPF fibroblasts to maintain a pathological phenotype on collagen by suppressing autophagy. However, the precise underlying mechanism by which the Akt downstream molecule suppresses autophagic activity remains elusive. FoxO3a is a direct target of Akt and is implicated with the transcriptional activation of autophagy. Therefore, we investigated whether reduced FoxO3a expression causes abnormally low autophagy in IPF fibroblasts on collagen. We found that FoxO3a mRNA and protein levels are low in IPF fibroblasts, which subsequently suppresses the autophagosomal marker LC3B expression on collagen matrix. In contrast, the majority of control fibroblasts showed an increase in FoxO3a and LC3B expression at both the mRNA and protein levels. The luciferase assay confirme...

Research paper thumbnail of Laser microsurgery in the GFP era: a cell biologist's perspective

Methods in cell biology, 2007

Modern biology is based largely on a reductionistic "dissection" approach-most cell bio... more Modern biology is based largely on a reductionistic "dissection" approach-most cell biologists try to determine how complex biological systems work by removing their individual parts and studying the effects of this removal on the system. A variety of enzymatic and mechanical methods have been developed to dissect large cell assemblies like tissues and organs. Further, individual proteins can be inactivated or removed within a cell by genetic manipulations (e.g., RNAi or gene knockouts). However, there is a growing demand for tools that allow intracellular manipulations at the level of individual organelles. Laser microsurgery is ideally suited for this purpose and the popularity of this approach is on the rise among cell biologists. In this chapter, we review some of the applications for laser microsurgery at the subcellular level and describe practical requirements for laser microsurgery instrumentation demanded in the field. We also outline a relatively inexpensive but ...

Research paper thumbnail of The spindle assembly checkpoint is satisfied in the absence of interkinetochore tension during mitosis with unreplicated genomes

The Journal of Cell Biology, 2008

The accuracy of chromosome segregation is enhanced by the spindle assembly checkpoint (SAC). The ... more The accuracy of chromosome segregation is enhanced by the spindle assembly checkpoint (SAC). The SAC is thought to monitor two distinct events: attachment of kinetochores to microtubules and the stretch of the centromere between the sister kinetochores that arises only when the chromosome becomes properly bioriented. We examined human cells undergoing mitosis with unreplicated genomes (MUG). Kinetochores in these cells are not paired, which implies that the centromere cannot be stretched; however, cells progress through mitosis. A SAC is present during MUG as cells arrest in response to nocodazole, taxol, or monastrol treatments. Mad2 is recruited to unattached MUG kinetochores and released upon their attachment. In contrast, BubR1 remains on attached kinetochores and exhibits a level of phosphorylation consistent with the inability of MUG spindles to establish normal levels of centromere tension. Thus, kinetochore attachment to microtubules is sufficient to satisfy the SAC even in ...

Research paper thumbnail of MicroRNA-96 inhibits FoxO3a function in IPF fibroblasts on type I collagen matrix

AJP: Lung Cellular and Molecular Physiology, 2014

Idiopathic pulmonary fibrosis (IPF) is a lethal and progressive lung disease characterized by per... more Idiopathic pulmonary fibrosis (IPF) is a lethal and progressive lung disease characterized by persistent (myo)fibroblasts and the relentless accumulation of collagen matrix. Unlike normal lung fibroblasts, IPF lung fibroblasts have suppressed forkhead box O3a (FoxO3a) activity, which allows them to expand in this diseased environment. microRNA-96 (miR-96) has recently been found to directly bind to the 3′-untranslated region of FoxO3a mRNA, which subsequently inhibits its function. We examined whether aberrantly low FoxO3a expression is in part due to increased miR-96 levels in IPF fibroblasts on polymerized collagen, thereby causing IPF fibroblasts to maintain their pathological properties. miR-96 expression was upregulated in IPF fibroblasts compared with control fibroblasts when cultured on collagen. In contrast, FoxO3a mRNA levels were reduced in most IPF fibroblasts. However, when miR-96 function was inhibited, FoxO3a mRNA and protein expression were increased, suppressing IPF ...

Research paper thumbnail of miR-210 promotes IPF fibroblast proliferation in response to hypoxia

American journal of physiology. Lung cellular and molecular physiology, Jan 15, 2014

Idiopathic pulmonary fibrosis (IPF) is characterized by the relentless spread of fibroblasts from... more Idiopathic pulmonary fibrosis (IPF) is characterized by the relentless spread of fibroblasts from scarred alveoli into adjacent alveolar units, resulting in progressive hypoxia and death by asphyxiation. Although hypoxia is a prominent clinical feature of IPF, the role of hypoxia as a driver of the progressive fibrotic nature of the disease has not been explored. Here, we demonstrate that hypoxia robustly stimulates the proliferation of IPF fibroblasts. We found that miR-210 expression markedly increases in IPF fibroblasts in response to hypoxia and that knockdown of miR-210 decreases hypoxia-induced IPF fibroblast proliferation. Silencing hypoxia-inducible factor (HIF)-2α inhibits the hypoxia-mediated increase in miR-210 expression and blocks IPF fibroblast proliferation, indicating that HIF-2α is upstream of miR-210. We demonstrate that the miR-210 downstream target MNT is repressed in hypoxic IPF fibroblasts and that knockdown of miR-210 increases MNT expression. Overexpression o...

Research paper thumbnail of Pathological Mesenchymal Progenitor Cells Reside In The Fibrotic Reticulum Of Idiopathic Pulmonary Fibrosis

B66. THE FIBROBLAST POOL ON THE MOVE: DERIVATION AND TARGETING, 2012

Research paper thumbnail of Pathologic Regulation of Collagen I by an Aberrant PP2A/HDAC4/miR-29 Signal Axis in IPF Fibroblasts

American Journal of Respiratory Cell and Molecular Biology, 2015

Idiopathic Pulmonary Fibrosis is characterized by the relentless expansion of fibroblasts deposit... more Idiopathic Pulmonary Fibrosis is characterized by the relentless expansion of fibroblasts depositing type I collagen within the alveolar wall and obliterating the alveolar airspace. miR-29 is a potent regulator of collagen expression. In IPF miR-29 levels are low while type I collagen expression is high. However the mechanism for suppression of miR-29 and increased type I collagen expression in IPF remains unclear. Here we show that when IPF fibroblasts are seeded on polymerized type I collagen, miR-29c levels are suppressed and type I collagen expression is high. In contrast, miR-29c is high and type I collagen expression is low in control fibroblasts. We demonstrate that the mechanism for suppression of miR-29 during IPF fibroblast interaction with polymerized collagen involves inappropriately low PP2A phosphatase function leading to HDAC4 phosphorylation and decreased nuclear translocation of HDAC4. We demonstrate that overexpression of HDAC4 in IPF fibroblasts restored miR-29c levels and decreased type I collagen expression, whereas knocking down HDAC4 in control fibroblasts suppressed miR-29c levels and increased type I collagen expression. Our data indicate that IPF fibroblast interaction with polymerized type I collagen results in an aberrant PP2A/HDAC4 axis which suppresses miR-29 causing a pathologic increase in type I collagen expression.

Research paper thumbnail of FoxO3a and disease progression

World Journal of Biological Chemistry, 2014

highlighted as a critical protein that regulates numerous cell functions from proliferation/apopt... more highlighted as a critical protein that regulates numerous cell functions from proliferation/apoptosis to stressresistance and aging. FoxO3a has been found to be deregulated in several diseases and FoxO3a targeting approaches are currently underway to treat various types of cancers. This review will describe the current concept of FoxO3a's pathological role in various diseases and elucidate the regulatory mechanisms involved. It will also provide the clinical significance and strategies to target FoxO3a to limit the progression of human diseases.

Research paper thumbnail of Transforming Growth Factor-β1 Induced Epithelial-Mesenchymal Transition in Hepatic Fibrosis

Hepatogastroenterology, 2011

The epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) are cruci... more The epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) are crucial for the regulation of cellular plasticity during liver fibrosis. Transforming growth factor (TGF)-β1 is an important cytokine for the induction of the EMT in liver fibrosis. TGF-β1 signaling induces the EMT through various signaling mechanisms and is the predominant agent mediating these fibrotic changes. Chronic exposure to TGF-β1 induces the transition of hepatocytes to collagen-producing mesenchymal cells, prolonged exposure of hepatocytes to TGF-β1 increases the expression of collagen and induces cytoskeletal rearrangement that resembles the EMT. These morphological and molecular alterations may provide the foundation for liver fibrosis. This review discussed the relation and mechanisms between EMT and liver fibrosis and ulteriorly elaborated on TGF-β1 induced EMT and each of their roles in liver fibrosis. Better understanding of the cellular and molecular characteristics of the cirrhotic hepatocyte may enable the development of chemo-preventative agents for liver fibrosis.

Research paper thumbnail of Identification of a Cell-of-Origin for Fibroblasts Comprising the Fibrotic Reticulum in Idiopathic Pulmonary Fibrosis

The American Journal of Pathology, 2014

Research paper thumbnail of Statistical Profiles of PTK1 Kinetochore Microtubule Plus-End Conformations Using High-Throughput Electron Tomography

Microscopy and Microanalysis, 2004

Extended abstract of a paper presented at Microscopy and Microanalysis 2004 in Savannah, Georgia,... more Extended abstract of a paper presented at Microscopy and Microanalysis 2004 in Savannah, Georgia, USA, August 1–5, 2004.

Research paper thumbnail of Low α2β1 Integrin Function Enhances the Proliferation of Fibroblasts from Patients with Idiopathic Pulmonary Fibrosis by Activation of the β-Catenin Pathway

The American Journal of Pathology, 2012

Idiopathic pulmonary fibrosis (IPF) is a progressive and incurable fibroproliferative disorder ch... more Idiopathic pulmonary fibrosis (IPF) is a progressive and incurable fibroproliferative disorder characterized by unrelenting proliferation of fibroblasts and their deposition of collagen within alveoli, resulting in permanently scarred, nonfunctional airspaces. Normally, polymerized collagen suppresses fibroblast proliferation and serves as a physiological restraint to limit fibroproliferation after tissue injury. The IPF fibroblast, however, is a pathologically altered cell that has acquired the capacity to elude the proliferation-suppressive effects of polymerized collagen. The mechanism for this phenomenon remains incompletely understood. Here, we demonstrate that expression of ␣ 2 ␤ 1 integrin, a major collagen receptor, is pathologically low in IPF fibroblasts interacting with polymerized collagen. Low integrin expression in IPF fibroblasts is associated with a failure to induce PP2A phosphatase activity, resulting in abnormally high levels of phosphorylated (inactive) GSK-3␤ and high levels of active ␤-catenin in the nucleus. Knockdown of ␤-catenin in IPF fibroblasts inhibits their ability to proliferate on collagen. Interdiction of ␣ 2 ␤ 1 integrin in control fibroblasts reproduces the IPF phenotype and leads to the inability of these cells to activate PP2A, resulting in high levels of phosphorylated GSK-3␤ and active ␤-catenin and in enhanced proliferation on collagen. Our findings indicate that the IPF fibroblast phenotype is characterized by low ␣ 2 ␤ 1 integrin expression, resulting in a failure of integrin to activate PP2A phosphatase, which permits inappropriate activation of the ␤-catenin pathway.

Research paper thumbnail of Pathological Alteration of FoxO3a Activity Promotes Idiopathic Pulmonary Fibrosis Fibroblast Proliferation on Type I Collagen Matrix

The American Journal of Pathology, 2011

Idiopathic pulmonary fibrosis (IPF) is a prevalent, progressive, and incurable fibroproliferative... more Idiopathic pulmonary fibrosis (IPF) is a prevalent, progressive, and incurable fibroproliferative lung disease. The phenotype of IPF fibroblasts is characterized by their ability to elude the proliferation-suppressive properties of polymerized type I collagen. The mechanism underlying this pathological response is incompletely understood but involves aberrant activation of the phosphatidylinositol 3-kinase-Akt signaling pathway owing to inappropriately low phosphatase and tensin homolog phosphatase activity. Akt can phosphorylate and inactivate the forkhead box O3a (FoxO3a) transcriptional factor, which, when transcriptionally active, increases the expression of the CDK inhibitor p27 and promotes cell cycle arrest. Herein, we demonstrate that IPF fibroblasts display high levels of inactive FoxO3a compared with nonfibrotic control fibroblasts because of their high Akt activity. We found that p27 levels are decreased in IPF compared with control fibroblasts cultured on polymerized collagen. Furthermore, overexpression of FoxO3a in IPF fibroblasts increases p27 levels and suppresses the ability of IPF fibroblasts to proliferate on polymerized collagen. In contrast, the expression of dominant-negative FoxO3a augmented control fibroblast proliferation. IHC examination of fibroblastic foci in IPF lung tissue demonstrates the presence of inactive FoxO3a in cells within fibroblastic foci. These data indicate that the ability of IPF fibroblasts to circumvent the proliferation-suppressive properties of polymerized collagen involves inactivation of FoxO3a by high Akt activity, resulting in down-regulation of p27.

Research paper thumbnail of Ultrastructure of Nocodazole-Treated PtK1 Kinetochore after High-Pressure Freezing and Freeze-Substitution

Extended abstract of a paper presented at Microscopy and Microanalysis 2005 in Honolulu, Hawaii, ... more Extended abstract of a paper presented at Microscopy and Microanalysis 2005 in Honolulu, Hawaii, USA, July 31--August 4, 2005

Research paper thumbnail of An insoluble protein matrix is required for centrosome function

Research paper thumbnail of The effects of collagen-rich extracellular matrix on the intracellular delivery of glycol chitosan nanoparticles in human lung fibroblasts

International Journal of Nanomedicine

Recent progress in nanomedicine has shown a strong possibility of targeted therapy for obstinate ... more Recent progress in nanomedicine has shown a strong possibility of targeted therapy for obstinate chronic lung diseases including idiopathic pulmonary fibrosis (IPF). IPF is a fatal lung disease characterized by persistent fibrotic fibroblasts in response to type I collagen-rich extracellular matrix. As a pathological microenvironment is important in understanding the biological behavior of nanoparticles, in vitro cellular uptake of glycol chitosan nanoparticles (CNPs) in human lung fibroblasts was comparatively studied in the presence or absence of type I collagen matrix. Primary human lung fibroblasts from non-IPF and IPF patients (n=6/group) showed significantly increased cellular uptake of CNPs (.33.6-78.1 times) when they were cultured on collagen matrix. To elucidate the underlying mechanism of enhanced cellular delivery of CNPs in lung fibroblasts on collagen, cells were pretreated with chlorpromazine, genistein, and amiloride to inhibit clathrin-mediated endocytosis, caveolae-mediated endocytosis, and macropinocytosis, respectively. Amiloride pretreatment remarkably reduced the cellular uptake of CNPs, suggesting that lung fibroblasts mainly utilize the macropinocytosisdependent mechanism when interacted with collagen. In addition, the internalization of CNPs was predominantly suppressed by a phosphoinositide 3-kinase (PI3K) inhibitor in IPF fibroblasts, indicating that enhanced PI3K activity associated with late-stage macropinocytosis can be particularly important for the enhanced cellular delivery of CNPs in IPF fibroblasts. Our study strongly supports the concept that a pathological microenvironment which surrounds lung fibroblasts has a significant impact on the intracellular delivery of nanoparticles. Based on the property of enhanced intracellular delivery of CNPs when fibroblasts are made to interact with a collagen-rich matrix, we suggest that CNPs may have great potential as a drug-carrier system for targeting fibrotic lung fibroblasts.

Research paper thumbnail of Idiopathic pulmonary fibrosis fibroblasts become resistant to Fas ligand-dependent apoptosis via the alteration of decoy receptor 3

The Journal of pathology, Sep 1, 2016

Idiopathic pulmonary fibrosis (IPF) is an irreversible lethal lung disease with an unknown etiolo... more Idiopathic pulmonary fibrosis (IPF) is an irreversible lethal lung disease with an unknown etiology. IPF patients' lung fibroblasts express inappropriately high Akt activity, protecting them in response to an apoptosis-inducing type I collagen matrix. FasL, a ligand for Fas, is known to be increased in the lung tissues of patients with IPF, implicated with the progression of IPF. Expression of Decoy Receptor3 (DcR3), which binds to FasL, thereby subsequently suppressing the FasL-Fas-dependent apoptotic pathway, is frequently altered in various human disease. However, the role of DcR3 in IPF fibroblasts in regulating their viability has not been examined. We found that enhanced DcR3 expression exists in the majority of IPF fibroblasts on collagen matrices, resulting in the protection of IPF fibroblasts from FasL-induced apoptosis. Abnormally high Akt activity suppresses GSK-3β function, thereby accumulating the nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) in the nuc...

Research paper thumbnail of LC3-2 expression was up-regulated in IPF fibroblasts in the presence of rapamycin on collagen

Research paper thumbnail of Caveolin-1 (Cav-1) Alteration By Aberrant Forkhead Box O3a (FoxO3a) Activity Confers A Highly Proliferative And An Anti-Apoptotic IPF Fibroblast Phenotypes On Type I Collagen Matrix

B66. THE FIBROBLAST POOL ON THE MOVE: DERIVATION AND TARGETING, 2012

Research paper thumbnail of Fibroblasts within the fibroblastic foci of IPF patient lung specimens express inactive FoxO3a and α-smooth muscle actin but not cav-1 and cleaved caspase-3

Research paper thumbnail of Reduced FoxO3a expression causes low autophagy in idiopathic pulmonary fibrosis fibroblasts on collagen matrices

American Journal of Physiology - Lung Cellular and Molecular Physiology, 2015

Idiopathic pulmonary fibrosis (IPF) is a chronic and fatal lung disease, and fibroblasts derived ... more Idiopathic pulmonary fibrosis (IPF) is a chronic and fatal lung disease, and fibroblasts derived from patients with IPF are resistant to type I collagen matrix-induced cell death. The alteration of the PTEN-Akt axis permits IPF fibroblasts to maintain a pathological phenotype on collagen by suppressing autophagy. However, the precise underlying mechanism by which the Akt downstream molecule suppresses autophagic activity remains elusive. FoxO3a is a direct target of Akt and is implicated with the transcriptional activation of autophagy. Therefore, we investigated whether reduced FoxO3a expression causes abnormally low autophagy in IPF fibroblasts on collagen. We found that FoxO3a mRNA and protein levels are low in IPF fibroblasts, which subsequently suppresses the autophagosomal marker LC3B expression on collagen matrix. In contrast, the majority of control fibroblasts showed an increase in FoxO3a and LC3B expression at both the mRNA and protein levels. The luciferase assay confirme...

Research paper thumbnail of Laser microsurgery in the GFP era: a cell biologist's perspective

Methods in cell biology, 2007

Modern biology is based largely on a reductionistic "dissection" approach-most cell bio... more Modern biology is based largely on a reductionistic "dissection" approach-most cell biologists try to determine how complex biological systems work by removing their individual parts and studying the effects of this removal on the system. A variety of enzymatic and mechanical methods have been developed to dissect large cell assemblies like tissues and organs. Further, individual proteins can be inactivated or removed within a cell by genetic manipulations (e.g., RNAi or gene knockouts). However, there is a growing demand for tools that allow intracellular manipulations at the level of individual organelles. Laser microsurgery is ideally suited for this purpose and the popularity of this approach is on the rise among cell biologists. In this chapter, we review some of the applications for laser microsurgery at the subcellular level and describe practical requirements for laser microsurgery instrumentation demanded in the field. We also outline a relatively inexpensive but ...

Research paper thumbnail of The spindle assembly checkpoint is satisfied in the absence of interkinetochore tension during mitosis with unreplicated genomes

The Journal of Cell Biology, 2008

The accuracy of chromosome segregation is enhanced by the spindle assembly checkpoint (SAC). The ... more The accuracy of chromosome segregation is enhanced by the spindle assembly checkpoint (SAC). The SAC is thought to monitor two distinct events: attachment of kinetochores to microtubules and the stretch of the centromere between the sister kinetochores that arises only when the chromosome becomes properly bioriented. We examined human cells undergoing mitosis with unreplicated genomes (MUG). Kinetochores in these cells are not paired, which implies that the centromere cannot be stretched; however, cells progress through mitosis. A SAC is present during MUG as cells arrest in response to nocodazole, taxol, or monastrol treatments. Mad2 is recruited to unattached MUG kinetochores and released upon their attachment. In contrast, BubR1 remains on attached kinetochores and exhibits a level of phosphorylation consistent with the inability of MUG spindles to establish normal levels of centromere tension. Thus, kinetochore attachment to microtubules is sufficient to satisfy the SAC even in ...

Research paper thumbnail of MicroRNA-96 inhibits FoxO3a function in IPF fibroblasts on type I collagen matrix

AJP: Lung Cellular and Molecular Physiology, 2014

Idiopathic pulmonary fibrosis (IPF) is a lethal and progressive lung disease characterized by per... more Idiopathic pulmonary fibrosis (IPF) is a lethal and progressive lung disease characterized by persistent (myo)fibroblasts and the relentless accumulation of collagen matrix. Unlike normal lung fibroblasts, IPF lung fibroblasts have suppressed forkhead box O3a (FoxO3a) activity, which allows them to expand in this diseased environment. microRNA-96 (miR-96) has recently been found to directly bind to the 3′-untranslated region of FoxO3a mRNA, which subsequently inhibits its function. We examined whether aberrantly low FoxO3a expression is in part due to increased miR-96 levels in IPF fibroblasts on polymerized collagen, thereby causing IPF fibroblasts to maintain their pathological properties. miR-96 expression was upregulated in IPF fibroblasts compared with control fibroblasts when cultured on collagen. In contrast, FoxO3a mRNA levels were reduced in most IPF fibroblasts. However, when miR-96 function was inhibited, FoxO3a mRNA and protein expression were increased, suppressing IPF ...

Research paper thumbnail of miR-210 promotes IPF fibroblast proliferation in response to hypoxia

American journal of physiology. Lung cellular and molecular physiology, Jan 15, 2014

Idiopathic pulmonary fibrosis (IPF) is characterized by the relentless spread of fibroblasts from... more Idiopathic pulmonary fibrosis (IPF) is characterized by the relentless spread of fibroblasts from scarred alveoli into adjacent alveolar units, resulting in progressive hypoxia and death by asphyxiation. Although hypoxia is a prominent clinical feature of IPF, the role of hypoxia as a driver of the progressive fibrotic nature of the disease has not been explored. Here, we demonstrate that hypoxia robustly stimulates the proliferation of IPF fibroblasts. We found that miR-210 expression markedly increases in IPF fibroblasts in response to hypoxia and that knockdown of miR-210 decreases hypoxia-induced IPF fibroblast proliferation. Silencing hypoxia-inducible factor (HIF)-2α inhibits the hypoxia-mediated increase in miR-210 expression and blocks IPF fibroblast proliferation, indicating that HIF-2α is upstream of miR-210. We demonstrate that the miR-210 downstream target MNT is repressed in hypoxic IPF fibroblasts and that knockdown of miR-210 increases MNT expression. Overexpression o...

Research paper thumbnail of Pathological Mesenchymal Progenitor Cells Reside In The Fibrotic Reticulum Of Idiopathic Pulmonary Fibrosis

B66. THE FIBROBLAST POOL ON THE MOVE: DERIVATION AND TARGETING, 2012

Research paper thumbnail of Pathologic Regulation of Collagen I by an Aberrant PP2A/HDAC4/miR-29 Signal Axis in IPF Fibroblasts

American Journal of Respiratory Cell and Molecular Biology, 2015

Idiopathic Pulmonary Fibrosis is characterized by the relentless expansion of fibroblasts deposit... more Idiopathic Pulmonary Fibrosis is characterized by the relentless expansion of fibroblasts depositing type I collagen within the alveolar wall and obliterating the alveolar airspace. miR-29 is a potent regulator of collagen expression. In IPF miR-29 levels are low while type I collagen expression is high. However the mechanism for suppression of miR-29 and increased type I collagen expression in IPF remains unclear. Here we show that when IPF fibroblasts are seeded on polymerized type I collagen, miR-29c levels are suppressed and type I collagen expression is high. In contrast, miR-29c is high and type I collagen expression is low in control fibroblasts. We demonstrate that the mechanism for suppression of miR-29 during IPF fibroblast interaction with polymerized collagen involves inappropriately low PP2A phosphatase function leading to HDAC4 phosphorylation and decreased nuclear translocation of HDAC4. We demonstrate that overexpression of HDAC4 in IPF fibroblasts restored miR-29c levels and decreased type I collagen expression, whereas knocking down HDAC4 in control fibroblasts suppressed miR-29c levels and increased type I collagen expression. Our data indicate that IPF fibroblast interaction with polymerized type I collagen results in an aberrant PP2A/HDAC4 axis which suppresses miR-29 causing a pathologic increase in type I collagen expression.

Research paper thumbnail of FoxO3a and disease progression

World Journal of Biological Chemistry, 2014

highlighted as a critical protein that regulates numerous cell functions from proliferation/apopt... more highlighted as a critical protein that regulates numerous cell functions from proliferation/apoptosis to stressresistance and aging. FoxO3a has been found to be deregulated in several diseases and FoxO3a targeting approaches are currently underway to treat various types of cancers. This review will describe the current concept of FoxO3a's pathological role in various diseases and elucidate the regulatory mechanisms involved. It will also provide the clinical significance and strategies to target FoxO3a to limit the progression of human diseases.